Process for the preparation of garamine and derivatives thereof

ABSTRACT

Amino-protected derivatives of garamine (useful intermediates) are prepared by treating a pseudotrisaccharide having a garamine moiety glycosidically linked to another pyranoside sugar possessing vicinal hydroxyl groups (preferably vicinal trans diequatorial hydroxyl groups), said pseudotrisaccharide having amino-protecting groups (preferably acetyl or benzoyl) with a glycol cleaving reagent (preferably sodium meta-periodate) and then treating the seco-dialdehyde derivative thereby formed with a phenylhydrazine and a lower alkanoic acid in water. Typical starting compounds include the per-N-acetyl derivatives of Antibiotic JI-20 Complex and the per-N-acetyl derivatives of a gentamicin mixture comprising gentamicins B, B1 and X2.

,United States Patent [1 1 Daniels 3,879,375 51 Apr. 22, 1975 [75] Inventor: Peter J. L. Daniels, Cedar Grove,

[73] Assignee: Schering Corporation, Bloomfield.

[22] Filed: June 26, 1973 v 211 App]. No.: 373,693

[52] U.S. Cl 260/210 R; 260/210 AB [51] Int. Cl. C07c 129/18 [58] Field of Search 260/210 R, 210 AB Primary E.\'aminer.lohnnie R. Brown Assistant E.\'aminerCary B. Owens Attorney, Agent, or Firm-Mary S. King; Stephen B. Coan I 57 1 ABSTRACT Amino-protected derivatives of garamine (useful intermediates) are prepared by treating a pseudotrisaccharide having a garamine moiety glycosidically linked to another pyranoside sugar possessing vicinal hydroxyl groups (preferably vicinal trans diequatorial hydroxyl groups), said pseudotrisaccharide having amino-protecting groups (preferably acetyl or benzoyl) with a glycol cleaving reagent (preferably sodium meta-periodate) and then treating the secodialdehyde derivative thereby formed with a phenylhydrazine and a lower alkanoic acid in water. Typical starting compounds include the per-N-acetyl derivatives of Antibiotic .lI-20 Complex and the per-N-acetyl derivatives of a gentamicin mixture comprising gentamicins B, B. and X 14 Claims, No Drawings PROCESS FOR THE PREPARATION OF GARAMINE AND DERIVATIVES THEREOF FIELD OF INVENTION This invention relates to a novel process and to novel intermediates useful therein.

More specifically. this invention relates to a novel process for preparing garamine and amino-protected derivatives thereof. such compounds being pseudodisaccharides useful as intermediates. from pseudotrisao charides. and to novel intermediates produced thereby.

In particular. this invention relates to the preparation of 1.3.3 '-tri-N-substituted garamine from a pseudotrisaccharide having a garamine moiety glycosidically linked to another pyranoside sugar possessing vicinal hydroxyl groups. said pseudotrisaccharide having amino-protecting groups. and to novel seco-dialdehyde intermediates produced thereby.

PRIOR ART Garamine. a pseudodisaccharide having antibacterial activity and also being useful as an intermediate. and l.3.3'-tri-N-substituted garamines. useful for producing pseudotrisaccharide antibacterial agents such as gentamicin X are described in co-pending application Ser. No. 296.434. filed Oct. It). 1972. and now abandoned of Alan K. Mallams for NOVEL INTERMEDI- ATES AND METHODS FOR PRODUCING GENTA- MICIN X The method described and claimed therein for preparing l.3.3'-tri-N-substituted garamine (and specifically 1.3.3 '-tri-N-carbobenzyloxygaramine) comprises selectively hydrolyzing by means of an acid. a pseudotrisaccharide having the garamine moiety and a hex-4-enopyranoside moiety (cg. sisomicin). said pseudotrisaccharide having N-protected groups (preferably carbobenzyloxy). The l.3.3.'-tri-N-substituted garamine thereby produced (e.g. l.3.3'-tri-N-car bobenzyloxygaramine) is then convertible to garamine via conventional deblocking methods. such as alkaline hydrolysis.

My invention provides an alternate method for preparing amino-protected garamine derivatives from different starting compounds. i.e. from pseudotrisaccharides having a garamine moiety glycosidically linked to another pyranoside sugar possessing vicinal hydroxyl groups. including known antibiotic pseudotrisaccharides such as Antibiotic G-4 l 8. gentamicin B. gentamicin B gentamicin X Antibiotics .II-ZOA and 11-208.

Additionally, by my process a mixture of pseudotrisaccharides having N-protecting groups may be conveniently converted together to produce an aminoprotected garamine derivative which is easily isolated from side reaction products and any remaining unreacted pseudotrisaccharide starting compound which did not have the requisite garamine moiety and pyranoside sugar bearing vicinal hydroxyl groups. Thus. my invention provides a method for preparing garamine derivatives which eliminates the necessity of extensive separation and purification of pseudotrisaccharide starting compounds from antibiotic complexes produced by fermentation.

My process utilizes a sequence of reactions similar to those known in the polysaccharide art as the Barry Degradation (OColla. P.S. The Barry Degradation; Mel/rods in Carbohydrate Chemistry, V pp. 382392. Academic Press, New York, 1965). However. prior to my invention, it was unknown to selectively degrade off a 2-amino sugar derivative from a polysaccharide via the Barry Degradation method.

GENERAL DESCRIPTION OF THE INVENTION In the process sought to be patented. a pseudotrisaccharide having a garamine moiety glycosidically linked to another pyranoside sugar which possesses vicinal hydroxyl groups. said pseudotrisaccharide having aminoprotecting groups. is treated with a glycol cleaving reagent whereby oxidative fission of the pyranoside sugar moiety occurs between the vicinal hydroxyl groups to produce a seco-dialdehyde derivative. which. upon treatment with a hydrazine selected from the group consisting of phenylhydrazine and a substituted phenyl hydrazine together with a lower alkanoic acid in water. produces a l.3.3'-tri-N-substituted garamine which. upon alkaline hydrolysis. is converted to garamine.

Preferred as starting compounds for my process are pseudotrisaccharides wherein the pyranoside sugar glycosidically linked to the garamine moiety possesses vicinal hydroxyl groups which are trans diequatorial to each other including aminoglycosides such as gentamicins B. B X Antibiotic G-4l8. Antibiotic Jl-ZUA. Antibiotic .lI-ZOB and mixtures thereof.

Glycol cleaving reagents useful in my process include lead (IV) salts. e.g. lead tetraacetate and phosphatolead(IV)-acids. and periodate salts. preferably sodium meta-periodate.

The hydrazine reagent preferably used in my process is phenylhydrazine. Other hydrazine reagents which may be used are phenylhydrazines substituted by groups such as methyl. nitro or halogen. including reagents such as p-bromophenylhydrazine. 2.4-dinitrophenylhydrazine. o-nitrophenylhydrazine. m-nitrophenylhydrazine. p-nitrophenylhydrazine. otolylhydrazine. m-tolylhydrazine. ptolylhydrazine. 2.3 xylylhydrazine. and 2.5-xylylhydrazine.

My process is usually carried out in a non-reactive solvent in which the starting pseudotrisaccharide and glycol cleaving reagent and hydrazine reagent are soluble. By non-reactive is meant a solvent which will not react with the pseudotrisaccharide or the reagents so as to cause transformations which will result in competing side reactions. When using a lead salt as cleaving reagent. my process is usually carried out in an organic sol vent such as lower alkanols and. preferably. lower alkanoic acids. When utilizing a periodate as cleaving reagent. water is the solvent of choice. although a lower alkanol may also be used.

The term amino-protecting groups as used in this specification and in the claims is well known in the art. Exemplifying such groups which are useful in my process are unsubstituted and functionally substituted acyl groups including lower alkanoyl groups such as acetyl and propionyl and aroyl groups such as benzoyl and Xyloyl. and aralkanoyl groups such as phenylacetyl. Amino-protecting groups particularly preferred for the process of this invention are acetyl and benzoyl.

Garamine and l.3.3'-N-substituted garamine. the pseudodisaccharides produced by my process. as described in copending application Ser. No. 296.434. filed Oct. 10. I972. may be represented by the following formula I:

. NHR

NHR

wherein R is an amino-protecting group or hydrogen. When R is hydrogen. the pseudodisaccharide having the chemical structure O-3-deoxy-4-C-methyl-3- methylamino-BL-arabinopyranosyl-( l o)-2-dexo v- D-streptamine as set forth in above formula I. has been named gar-amine. which compound possesses anti bacterial activity per se. being useful in treating conditions caused by S. aurciis'. E. (u/i. P. uerugiliusu and other pathogenic organisms.

Compounds of formula I wherein R is an aminoprotecting group (preferably acetyl or benzoyl in the process of this invention) are termed amino-protected derivatives ofgaramine and are the compounds directly produced by my process. These derivatives may be methyl. R is amino. X is hydroxyl). and gentamicin X (also known in the art as gentamicin X) (a compound y of formula II wherein R and R are hydrogen. R is hyused as is as intermediates for preparing trisaccharides dmxyi and X iS amino); and Antibiotics A and utilizing either the Koenigs-Knorr or the Lemieux reac- 20B (R iS hydrogen Rl iS hydrogen and methyl, respec tions with a monosaccharide as the other reagent. For iii-fly R9 and X are Cuch amino) which are described example. gentamicin X a known antibacterial agent. in copendiiig application Serial 261 753-fiied June may be prepared from l.3.3.'-tri-N-acetylgaramine and 13 972 f j |iavski i p Bayan, wiui (h the monosaccharide 3.4.o-tri-O-acetyI-Z-deoxy-Znimy and Hans Reimunn f NEW ANTIBIOTIC FROM trosocx-D-glucopyranosyl chloride via the well-known MICROMONOSPORA PURPUREA 2() Lemieux procedure followed by reduction and removal Carrying out m process whereby amino-protected of the hloc king p Ahemillivel. pseudotrisaccharides represented by formula II are de acetylgaraminc may be COI'IVCI'ItId l0 O-protected (it)- 25 graded to pseudodisagcharides represented by formula rivativcs thereof also useful in the synthesis of antibac' Le, to aminorote ted garamine derivatives. a lfiriillspseudotrisaccharide derivative represented by formula I Y! i I Additionally. amino-protected garaniine derivatives 311 l' l l 'g i fi n f f 5 1 a of formula I prepared according to the process of this in g fi i 2 F f I T invention. e.g. l.3.3'-tri-N-acetylgaramine and 2 d Fi dcetiy i i fi fi l.3.3'-tri-N-benioylgaramine. may be deblocked via f i quaint) i i? C i i alkaline hvdrolvsis and the garamine therebv produced redgen pre cm f m er r room temperature until thin layer chromatographic may be converted utilizing knonn techniques to ananalvsis of an aliquot of the reaction solution indicates other amino-protected dern ative. e.g. to the 1.3.3 -mthe absence of starting compound. When a periodate lS N-carbobenzyloxygaramine. which may be more desirv employed as reagent. the excess periodate and morable for use in a given sequence of reactions.

ganic salts are conveniently removed by precipitation Particularly useful amino-protected pseudotrisacchawith lead to acetate and. after removing the resultant ride starting compounds for the process of this invenprecipitate by filtration. then removing further inortion are aminoglycosides represented by the following 40 ganic ions by bringing the aqueous filtrate to about pl l formula II and mixtures thereof: by means of dilute sulfuric acid. and filtering off the re- NHR wwm MR2 u R H o H his e t wherein R is an amino-protecting group as defined sulting precipitated inorganic salts. After removal of hereinabove. preferably acetyl or benzoyl; R is a memthe periodate and other inorganic salts. there remains ber selected from the group consisting of hydrogen and an aqueous solution of the sccodialdehyde oxidation methyl; R and X are each a member selected from the product which may be represented by the following formula lll:

WCWR

CHO

NHR

RHR

III

wherein R. R and R are as defined in formula II and Y is a member selected from the group consisting of CH0 and M -CHCHO.

In the conversion of the pseudotrisaccharide (ll) to the seco-dialdehyde oxidation product (lll). there occurs oxidative fission of the original pyranoside sugar moiety between the vicinal hydroxyl groups. Thus. when a starting pseudotrisaccharide possesses three vicinal hydroxyl groups. e.g. compounds of formula ll wherein X is hydroxyl such as in gentamicins B and 8., fission occurs at two places to produce a secodialdehyde offormula lll wherein Y is CHO together with formic acid.

ln carrying out the process of this invention. it is not necessary to isolate or purify the seco-dialdehyde intermediate lll. and the aqueous reaction solution containing the seco-dialdehyde oxidation product is usually immediately treated with a hydrazine reagent. preferably phenylhydrazine. and aqueous acetic acid at temperatures in the range of from about 45 to about l00C (usually at reflux temperature) until thin layer chromatographic analysis of an aliquot of the reaction solution indicates the absence of starting compound and the presence of mainly l.3.3'-tri-N-substituted garamine. Isolation of the resulting garamine derivative of formula I is conveniently effected by concentrating the reaction mixture to a small volume. dissolving the residue in a week base (e.g. ZN-ammonium hydroxide) extracting the basic aqueous mixture with an organic solvent (preferably a halogenated hydrocarbon solvent such as chloroform). and concentrating the aqueous solution to a residue comprising a l.3.3'-tri-N- substituted garamine of formula I which may be purified utilizing known techniques such as extraction. crystallization and chromatography.

When garamine is desired. the amino-protected garamine derivative of formula I may be deblocked by known methods. preferably. alkaline hydrolysis.

The seco-dialdehyde intermediate represented by formula III as set forth hereinabove may exist in one or more acetal or hydrated acetal forms; accordingly, formula III is placed in brackets and it is to be understood that in this specification and in the claims. the term seco-dialdehyde intermediate of formula ll includes the seco-dialdehyde structure set forth in formula III and any equivalent acetal or hydrated acetal form thereof.

My process may be carried out utilizing a mixture of pseudotrisaccharides comprising aminoglycosides possessing the requisite garamine and other pyranoside sugar having vicinal hydroxyl groups. as well as other compoundsnot possessing the requisite groups. and there will be produced l.3.3'-tri-N-substituted garamine which can be easily separated from other compounds via chromatographic techniques. For example. a mixture of pseudotrisaccharides obtained as described in J. Chrmnamgmp/zy 70. 171 (1972) by removal of gentamicins A and C from the total antibiotic complex produced in the gentamicin fermentation by Mir'rmnonspom purpureu NRR] 2953 (said mixture comprising gentamicins B. B, and X together with small quantities of gentamicins A and C] upon conversion to a per-amino-protected mixture (e.g. to a per-N- acetyl pseudotrisaccharide mixture comprising 1.3.6,- 3"-tetra-N-acetylgentamicin B. l.3.6',3"-tetra-N- acetylgentamicin 8,, 1.3.2 ',3 '-tetra-N- acetylgentamicin X together with small quantities of l.3.2',3"-tetra-N-acetylgentamicin A and l.3,2',6",3- "-penta-N-acetylgentamicin C) and treatment of the N-protected mixture according to the process of this invention. yields l.3,3'-tri-N-substituted garamine (e.g. l.3.3-tri-N-acetylgaramine) which is easily separated via chromatographic techniques from side products and residual nonreacting starting pseudotrisaccharides such as gentamicin C.

Similarly. Antibiotic Jl-ZO Complex (comprising Jl 20A and .ll-ZOB) which is produced in the fermentation of Micrumunospom pm'purea Jl-ZU (NRRL 5467) and isolated as described in the Preparation and Examples of copending Application U.S. Ser. No. 261.753 filed June [2. l972. upon conversion to the per-N-acetyl derivative thereof (i.e. to l.3.2',6',3"-penta-N- acetylantibiotic JI-20 Complex) and thence treatment with sodium meta-periodate followed by treatment of the seco-dialdehyde intermediate thereby produced with phenylhydrazine and acetic acid yields l.3.3"tri- N-acetylgaramine.

My process is described in detail for aminoglycoside derivatives having acetyl-N-protecting groups which is 7 a preferred amino protecting group when carrying out the process of this inventionv It is to be understood. however. that other lower alkanoyl groups. e .g. propionyl. and aroyl groups. e.g. benzoyl. are also prefcri'ed blocking groups for my process.

Procedures are set forth hereinbelow to illustrate the best mode contemplated by applicant for carrying out my invention and are not to be construed as limiting the scope thereof.

PREPARATION l Per-N-Lower AlkanoyIaminoglycosides A. l.3.2'.3"-Tetra-N-Acetylantibiotic -418 To Antibiotic (3-418 (20 g.) in methanol (100 ml.) add acetic anhydride 1111.]. then allow the reaction mixture to stand at room temperature for 2.5 hours. Concentrate the solution in vacuo. redissolve the resultant residue in methanol ml.). and add the methanol solution dropwise to 1 liter of anhydrous ether. Separate by filtration the resultant precipitate to obtain 2.5 g. of l.3.2'.3"-tetra-N-acetylantibiotic G-4I8 as a colorless solid. which. upon thin layer chromatographic analysis on silica ge. G.F. utilizing chloroformzmcthanolcammonium hydroxide 1:1 :1 as developing solvent shows a single spot more mobile than Antibiotic 6-418v In a manner similar to that described in Preparation 1A treat each of the following aminoglycosides with acetic anhydride in methanol: gcntamicin B. gentamicin B gentamicin X and a mixture comprising the foregoing together with small quantities of gentamicins A and C and other minor components (prepared as described in J. ('lironmmgr. Volume 70. 171-173. I972).

Isolate and purify each of the resulting products in a manner similar to that described in Example 1A. to obtain. respectively:

I.3.6'.3"-tetra-N-acetylgentamicin B as the tetrahydrate; [111 ,26 119 (water).

1.3.6',3"-tetra-N-acetylgentamicin B, as the trihydrate. [011 ,26 134 (water).

1.3.2,3"-tetra-N-acetylgentamicin X and an acctylated gentamicin mixture comprising 1.3.6.- 3"-tctra-N-acctylgentamicin B: l.3.6'.3"-tetra-N- acetylgentamicin B,'. and I.3.2',3"-tetra-N- acetylgentamicin X C. I.3.2'.6'.3"-Penta-N-Acetylantibiotics JI-20B. and 11-20 Complex The requisite starting compounds i.e. Antibiotics JI- 20A. 11-208. and 1120 Complex are prepared as described in Examples 1-3 of copending application U.S. Ser. No. 261.753 filed June 12. 1972 and now abandoned.

1. Add 4 ml. ofacetic anhydride dropwise to a stirred solution of I gm. of Antibiotic Jl-20A in ml. of methanol at 5C. Stir the reaction mixture at room temperature for 16 hours. then concentrate to about one half volume in vacuo and add dropwise to 50 ml. of stirred diethyl ether. Isolate the resultant precipitate by filtration. dissolve the precipitate in a minimum volume of aqueous methanol and chromatograph on'a column of silica gel. Elute with the lower phase ofa 1:1:1 chloroform: methanolzammonium hydroxide solvent system. Monitor fractions by thin layer chromatography. combine those fractions containing penta-N- acetylantibiotic JI-20A, then concentrate the combined fractions to a residue. Dissolve the residue in water. filter. and Iyophilize to obtain l.3.2',6.3"-penta- JI-2OA.

. 8 N-acetylantibiotic Jl-20Ai m.p. 210-220C1 [01],,26 141(water).

2. In similar manner. test each of Antibiotic 11-208 and Antibiotic 11-20 complex (comprising Antibiotic .II-20A. Antibiotic JI-20B and smaller amounts of gentamicin C,. C: and C,,,) in methanol and acetic anhydride at room temperature for 16 hours. Isolate each product by filtering the respective reaction solution and pouring each filtrate into stirred ethyl ether. Collect each of the resultant precipitates by filtration, wash with ether and dry in vacuo to obtain respectively:

l.3.2'.6'.3"-penta-N-acetylantibiotic Jl-20B: m.p. 220225C; [a],,26 124 (water); and

l.3.2.'.60'.3"-penta-N-acetylantibiotic .lI-20 complex: m.p. 216224C; lalnlb (water).

D. In the procedure of Preparations 1A. 1B. and 1C. by substituting other lower alkanoic acid anhydrides (e.g. propionic acid anhydride and caprylic acid anhydride) for acetic acid anhydride. there is formed the corresonding per-N-alkanoyl derivativev e.g. the corresponding per-N-propionyl and per-N-capryloyl derivatives of the compounds of Preparation 1A. 1B. and 1C respectively.

PREPARATION 2 Per-N-Benzoylaminoglycosides A. l.3.6,3"-tetra-N-Benzoylgentamicin B l. Dissolve 4.82 g. ofgentamicin B in 250 ml. ofdried pyridine. and cool to 0C. Add with stirring 21.1 g. of benzoyl chloride. and allow the 'reaction mixture to stand at room temperature for 24 hours. Evaporate the pyridine in vacuo. dissolve the resultant residue in 500 ml. of chloroform. wash the chloroform solution with aqueous sodium bicarbonate solution. 0.1N sulfuric acid and then saturated sodium chloride solution. dry the washed chloroform solution over sodium sulfate, filter. and evaporate to a resiude. Dissolve the residue in 500 ml. of methanol. treat with a small quantity of sodium methoxide and allow to stand at room temperature for 24 hours. Evaporate the methanol solution in vacuo to a residue. then redissolve the residue in a minimum volume of methanol and add the methanolic solution to a large volume of stirred ether. Filter and dry the resultant precipitate comprising 1.3.6',3"-tetra-N- benzoylgentamicin B.

B. In a similar inanner. treat each of the following with benzoyl chloride a pyridine at 0C followed by sodium methoxide in methanol: gentamicin 8,, gentamicin X a mixture comprising gentamicins B. B, and X Antibiotic G4I8. Antibiotic JI-20A. Antibiotic JI- ZOB. and Antibiotic 11-20 complex.

Isolate and purify each of the resultant products in a manner similar to that described hereinabove to obtain respectively 1.3.6',3"-tetra-N-benzoylgentamicin B l,3,2'.3"-tetra-N-benzoylgentamicin X a product mixture comprising tetra-N-benzoylgentamicins B. B and X 1.3.2',3"-tetra-N-benzoylantibiotic (3-418; 1.- 3.2.6',3"-penta-N-benzoylantibiotic Jl-20A'. 1.32.6- .3"-penta-N-benzoylantibiotic Jl-20B; 1.3.2'.6',3"- penta-N-benzoylantibiotic 11-20 complex.

C. In the procedures of Preparations 2A and 28. by substituting other aroyl halides. e.g. p-toluoyl bromide and xyloyl chloride. for benzoyl chloride. there is formed the corresponding per-N-aroyl derivative. eg the corresponding per-N-toluoyl and the corresponding per-N-xyloyl derivatives of the compounds of Preparations 2A and 2B.

To a stirred solution of l g. of l.3.2',3"-tetra-N- acetylantibiotic G-4l8 in 100 ml. of'water. add 1 equivalent of sodium meta-periodate (0.321 g.). Stir the so lution at room temperature overnight or until thin layer chromatographic analysis of an aliquot of the reaction solution on silica gel G. F. with chloroformzmethanol- :ammonium hydroxide (l:l:l) as developing solvent shows the absence of starting material. To the reaction solution. add dropwise 5 ml. of a percent aqueous solution of lead diacetate. Remove the resultant inorganic precipitate by filtration. then treat the resultant filtrate with 0. lN sulfuric acid ml.) until the solution is at about ph 4. Separate by filtration the resultant inorganic precipitate to obtain an aqueous solution containing the seco-dialdehyde derivative of tetra-N- acetylantibiotic (3-418. represented by the following formula lA:

' OHC IHCOCHg NHCOCB HCOCH;

hydroxide. then extract the aqueous solution with three ml. portions of chloroform. Concentrate the washed aqueous layer in vacuo to a residue comprising l 3.3 -tri-N-acetylgaramine.

Purify by column chromatography (2.5 by cm.) utilizing g. of silica gel previously conditioned by contact with the lower phase of a solvent mixture comprising chloroform: methanolzammonium hydroxide (2:111 Dissolve the tri-N-acetylgaramine prepared as described above in a minimum volume of the lower phase of the solvent mixture comprising chloroform:methanol:ammonium hydroxide (2:1:l) and place this solution on the silica gel column. eluting with chloroform:methanolzammonium hydroxide (2: l :1 Monitor the eluates by thin layer chromatography on silica plates (250 microns). developing by spraying with 5 percent sulfuric acid in methanol and charring. Combine like fractions comprising pure l.3 3'-tri-N-acetylgaramine and evaporate the combined fractions in vacuo to a residue comprising l.33'-tri-N-acetylgaramine: [a],,26 l02.9 (0.3% in methanol); yield 264 mg.

EXAMPLE 2 Preparation of l.3.3' tri-N-acetylgaramine From Per-N-Acetylgentamicin B A. Periodate Oxidation of Tetra-N-Acetylgentamicin B To l,3.6,3"-tetra-.N-acetylgentamicin B mg. 0.3 mM) add a solution of sodium meta-periodate (99 mg.. 0.462mM) in 20 ml. of water. Stir at room temper- 5 ature until thin layer chromatographic analysis of an i aliquot of the reaction solution indicates the absence of starting material. Add 30 ml. of a 1 percent solution of lead diacetate to the reaction mixture with stirring. Separate the resultant precipitate by filtration and cautiously add dropwise 2 ml. of 4 percent sulfuric acid.

EXAMPLE 3 The Preparation of l.3.3-tri-N-acetylgaramine from per-N-acetylgentamicin B, A. In a manner similar to that described in Example Separate the resultant precipitate of lead sulfate by fi]- 5 2A. treat [50 mg. of l. .6'. et fltration and evaporate the aqueous filtrate to a residue llcelylgemilmlcin 1 with 100 Of Sodium mall (2| 1.5 mg.) comprising the tetra-N-acetvlgentamicin B pcnodatej :01am and punfy the resultant pmducl in a seco-dialdehyde product represented by the following F similar to that descnbed to i i u Soluuon 1 I t I f 1. 3A containing the tetra-N-acetylgentam1cm B secoruL um dialdehyde oxidation product represented by the fol- 101C968; HENHCOCHS NHCOCH nmmww-L B. l.3.3'-Tri N-Acetylgaramine lowing structural formula 3A wherein Z represents the Dissolve the 21 1.5 mg. of oxidized product obtained 4-0-linkedper-N-acetylgaramine residue: in Example 2A in 25 ml. of water and add 1 ml. of phenylhydrazine and 1 ml. of acetic acid. Heat at reflux temperature for 1 hour or until thin layer chromatographic analysis on silica gel indicates the absence of CH3 RHCOCH: starting material. Allow the reaction mlxture to stand so overnight at 25C. Evaporate in vacuo to a residue. dis 0 solve the residue in 2N ammonium hydroxide ml.). g

. HO I extract the aqueous solution with two 50 ml. portions Z of chloroform. and evaporate the extracted aqueous layer to a residue comprising l,3.3'-tri-N- 5A acetylgaramlne. Purify by dissolving the trl-N- I acetylga amine in methanol and treating with charcoal B. in a manner similar 0 [hill dCSCl'lbCd [11 Example at room temperature for one hour. then at reflux tem- 2B, treat the solution of tetra-N-acetylgentamrc1n B perature for five minutes. Filter. evaporate the methadialdehyde oxidation product obtained as described nolic solution to a residue. redissolve the residue in 0.5 above with phenylhydrazine and acetic acid in water.

ml. of methanol. then add dropwise l5 ml. of ether. Separate by filtration the resultant precipitate'comprising l.3.3'-tri-N-acetylgaramine (52.6 mg.. 50.8% yield) [011 ,26 100.3 (0.3% in methanol).

Isolate the resultant product in a manner similar to that described to obtain l.3.3-tri-N-acetylgaramine.

Purify by chromatographing over silica gel (8 g.)

eluting with the lower phase of a chloroform:methanol- :concentrated ammonium hydroxide system. Combine like fractions as determined by thin layer chromatography. then evaporate the combined fractions to obtain l.33'-tri-Nacetylgaramine (23.5 mg.. 23% theory).

EXAMPLE 4 Preparation of l,3.3'-tri-N-acetylgaramine from Per-N-Acetylgentamicin X:

CHgOI'l Z OHC NRC QC H B. l3,3-Tri-N-Acetylgaramine In a manner similar to that described in Example 18. treat the aqueous filtrate obtained in Example 4A with phenyl hydrazine. acetic acid and water. Isolate and purify the resultant product in a manner similar to that described to obtain l3.3'-tri-N-acetylgaramine.

EXAMPLE 5 Preparation of l.3.3-Tri-N-acetylgaramine from a Mixture Comprising Tetra-N-acetylgentamicin B. tetra-N-acetylgentamicin B,. and Tetra-N-acetylgentamicin X A. To a stirred solution of 17 g. of an N acetylated gentamicin mixture (prepared as described in Preparation 18) in 250 ml. of water, add g. of sodium metaperiodate. Stir the solution overnight. then add a It) percent aqueous solution of lead diacetate dropwise until no additional precipitate appears. Separate the re sulting inorganic precipitation by filtration, then add 0.1N sulfuric acid until the filtrate is at pH 4-4.5. Separate the resultant precipitate of lead sulfate by filtration to obtain a filtrate containing a product mixture comprising the seco-dialdehyde derivative of gentamicins B, B and X B. To the solution of the seco-dialdehyde product mixture obtained in above Example 5A. add I [5 ml. of phenylhydrazine, 115 ml. of glacial acetic acid. then water, until the total volume of the reaction solution is 2 liters. Heat the reaction mixture at reflux temperature for 1 hour. Concentrate the reaction mixture to a syrupy residue. dissolve the residue in two liters of 2N- ammonium hydroxide, extract the ammonium hydroxide solution with three 280 ml. portions of chloroform. then concentrate the aqueous layer to a residue comprising l.3.3-tri-N-acetylgaramine. Chromatograph the residue on a 215 by 13 cm. column filled with 400 g. of silica gel previously conditioned by contact with the lower phase of a solvent mixture comprising chloroform:methanoliammonium hydroxide (2: l :l 1. Dissolve the l.3.3'-tri-N-acetylgaramine prepared as described in a minimum volume of the same chloroform:methanol:ammonium hydroxide (2:1:l solvent mixture. pour the solution on the column. and elute with the same solvent mixture at a flow rate of 2 ml. per minute. Monitor the effluent by thin layer chromatography. Combine like fractions containing pure tri-N-acetylgaramine. and evaporate the combined eluates to a residue comprising 8.9 g. of l.3.3'-tri-N- acetylgaramine.

EXAMPLE 6 Preparation of Tri-N-acetylgaramine from Antibiotics JI-ZOA. JI-ZOB. and .Il-ZU Complex A. The Periodate Oxidation of penta-N- acetylantibiotics Jl-ZOA. Jl-ZOB. and .ll-20 Complex 1. In a manner similar to that described in Example lA. treat an aqueous solution of 1 gm. of l.3,2',6'.3"- penta-N-acetylantibiotic .II-ZOA in I00 ml. of water with 1 equivalent of sodium meta-periodate. Stir the solution at room temperature until thin layer chromatographic analysis indicates the absence of starting material. Treat the reaction solution with 10 percent aqueous lead diacetate. then 0. IN sulfuric acid. and remove the resultant inorganic precipitate by filtration to obtain an aqueous solution containing the penta-N- acetylantibiotic-Jl-ZUA seco-dialdehyde oxidation product represented by the following formula 6A( 1) wherein Z is a 4-O-linked-tri-N-acetylgaramine residue:

ca mieoca use . one

uncocaa 2. In similar manner. treat l,3.2',6',3"-penta-N- acctylantibiotic .ll-ZUB with sodium periodate in water. followed by treatment of the reaction mixture with aqueous lead diacetate, then 0. IN sulfuric acid. Isolate the resultant product in a manner similar to that described in above Example 6A( 1) to obtain an aqueous solution containing penta-N-acetylantibiotic JI-ZOB di- WD W-NOCHCH I DEC mrcocrr 34 In similar manner. treat penta-N-acetylantibiotic Jl-20 complex in water with sodium meta-periodate followed by percent aqueous solution of lead diacetate. then 0.1N sulfuric acid. Remove the resultant pre cipitate of lead sulfate by filtration to obtain an aqueous solution containing a seco-dialdehyde product mixture comprising the compounds of Examples 6A(l) and 6A(2).

B. l.3.3'-Tri-N-acctylgaramine Treat each of the aqueous filtrates obtained in Examples 6A( l 6A(2) and 6A(3) containing the secodiuldehyde derivatives of per-N-acetylantibiotics 1]- A. Jl-ZOB. and .ll-20 complex. respectively, with phenyl hydrazine in acetic acid and water in a manner similar to that described in Example 13. Isolate and purify the resultant product in a manner similar to that described to obtain l.3.3'-tri-N-acetylgaramine.

EXAMPLE 7 Preparation of I33 '-tri-N-Benzoylgaramine /l o CHO 1 In a manner similar to that described in Example 28, treat the seco-dialdehyde derivative of tetra-N-benzoylgentamicin B dissolved in water with phenyl hydrazinc and acetic acid. Isolate and purify the resultant product in a manner similar to that described to obtain 1.3,3 -tri-N-benzoylgaramine.

In similar manner, subject the following per-N- benzoyl derivatives to the procedures of Example 7A and 78 to obtain l3.3'-triN-benzoylgaramine:

l.3.6',3"-tetra-N-benzoylgentamicin B,;

l 3.2',3"-tetra-N-benzoylgentamicin X a mixture of tetra-N-benzoylgentamicin B. B and X 13.2, 3"-tetra-N-benzoylantibiotic 0-418;

l.3.2,6"-penta-N-benzoylantibiotic .ll-ZOA;

l,3.2'.6"-penta-N-benzoylantibiotic JLZOB and mixtures thereof: and l.32',6,3"-penta-N-benzoylantibiotic .ll-ZU complex.

EXAMPLE 8 GARAMINE 1. Heat 500 mg. of l,3.3'-tri-N-acetylgaramine in 25 for 96 hours. Pass the solution through a column of [RC- resin (NH cycle). Wash the column with two column volumes of water followed by 0. IN ammonium hydroxide. Monitor the fractions by thin layer chromatography. combine the like fractions containing garamine, and evaporate to a residue comprising garamine.

In similar manner, treat 1,3,3 '-tri-N benzoylgaramine and l.3,3'-tri-N-carbobenzyloxygaramine with aqueous sodium hydroxide and isolate and purify the resultant product as described hereinabove to obtain garamine.

I claim:

1. The process for preparing a garamine derivative having amino-protecting groups selected from the group consisting of lower alkanoyl, aroyl and aralkanoyl. which comprises treating a pseudotrisaccharide having a garamine moiety 4-Q-glycosidically linked to another pyranoside sugar which possesses an amino function and vicinal hydroxyl groups, said pseudotrisaccharide having amino-protecting groups selected from the group consisting of lower alkanoyl, aroyl and aralkanoyl, with a glycol cleaving reagent selected from the group consisting of a periodate salt and a lead (IV) salt; and treating the seco-dialdehyde intermediate with a hydrazine and a lower. alkanoic acid in water, said hydrazine being a phenylhydrazine.

2. The process of claim 1 wherein said pseudotrisaccharide has a garamine moiety 4-Q-glycosidieally linked to another pyranoside sugar with possesses an amino function and vicinal trans diequatorial hydroxyl groups, and wherein said pseudotrisaccharide has amino-protecting groups selected from the group consisting of lower alkanoyl and aroyl having up to 8 can bon atoms.

3. The process of claim 2 followed by the step of treating the garamine derivative with alkali whereby garamine is formed.

4. The process of claim 2 for preparing an aminoprotected garamine derivative represented by the following structural formula I:

III-1R wherein R is an amino-protecting group selected from the group consisting of lower alkanoyl and aroyl having up to 8 carbon atoms; which comprises treating a pseudotrisaccharide selected from the group consisting ofa compound represented by the following formula II and mixtures thereof:

NHR

wherein R is as hereinabove defined; R, is a member selected from the group consisting of hydrogen and methyl, R and X are each a member selected from the group consisting of NHR and hydroxy;

with a glycol cleaving reagent selected from the group consisting of a periodate salt and a lead salt; and treating the seco-dialdehyde intermediate selected from the group consisting of a compound represented by the following structural formula III and mixtures thereof:

NHR

NHR

wherein R. R and R are as hereinabove defined. and Y 15 a member selected from the group consisting of -C=0 and HCHO;

with a hydrazine and a lower alkanoic acid in water. said hydrazine being a phenylhydrazine.

5. The process of claim 4 wherein R is benzoyl.

6. The process of claim 4 wherein R is acetyl.

7. The process of claim 4 wherein said glycol cleaving reagent is an alkali metal meta-periodate. said hydrazine is phenylhydrazine. and said lower alkanoic acid is acetic acid 8. The process of claim 4 wherein said pscudotrisac charide is a compound of formula ll or a mixture thereof. wherein R is acetyl; said glycol cleaving reagent is sodium meta-periodate; said hydrazine is phenylhydrazine and said lower alkanoic acid is acetic acid whereby is formed l.3,3'-tri-N-acetylgaramine.

9. The process of claim 8 wherein said pseudotrisaccharide represented by formula ll is l.3r2.3"-tetra-N- acetylantibiotic G-4l 8.

10. The process of claim 8 wherein said pseudotrisaccharide represented by formula ll is l.3 6,3"-tetra-N- acetylgentamicin B 11. The process of claim 8 wherein said pseudotrisaccharide starting compound is a mixture comprising 1.3- .2',6',3"-penta-N-acetylantibiotic Jl-20A and l.3,2',6- ,3"-penta-N-acetylantibiotic 11-208.

12. The process of claim 8 wherein said pseudotrisac charide represented by formula ll is l,3 2',6',3"-penta- N-acetylantibiotic Jl-ZOB.

13. The process of claim 8 wherein said pseudotrisaccharide starting compound is a mixture comprising 1.3- ,6',3"-tetra-N-acetylgentamicin B, l,3 6',3"-tetra-N- acetylgentamicin B and 1,3 2 ',3 '-tetra-N- acetylgentamicin X 14. The process of claim 8 followed by the steps of treating the l .3 3-tri-N-acetylgaramine with alkali and isolating garamine.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION PATENT NO. 5,879, 575 DATED April 22, 1975 IN 1 Peter J. L. Daniels It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

. Column line 58, "with lead to acetate and," should read ---with lead acetate and,---. Column line +0,' "to about pH should read ---to about pH Column 6, line 2 "of formula II" should read ---of formula III"--- Column 6, line 40, "NRRI 2955" should read ---NRRL 2955---. Column 6, lines 1-8 and +9, "and l, 5,2 ,6", 5" should read ---and l, 5,,

. 2' ,6' ,5"- Column 7, line 25, "on silica ge" should read ---on silica gel--- Column 7, line 59, OI 7 26 should read [or i Column 7, line rl, "[aJ 26" should read ---[Ot 7% Column 8, line 1, "[0t 7 26 should read 7 Column 8, line 15, Ot 7 26 should read O! 7 Column 8, lines l r and 15, "1,5,2', D 60' 5" 05 7 26 130 (water)" should read l, 5,2 ,6' 5"- [or 7 150 (water)---. Column 8, line 4-8, "chloride a pyrid ne" should read ---chloride in pyridine---. Column 9, line 18, "about ph r" should read ---about pH Column 9, line 59, "the solution of reflux" should read ---the solution at refluX---. Column 10, line 22, "[orJ 26 rr should read Q 273 Column 11, line 6 "[OLT 26 should read [oz 7; Column 16, line 10, "l, 3,2' ,6"-penta" should read ---l,5,2',6'-penta---. Column 16, line 11, "l,5,2',6"-penta' should read ---l,5,2',6',5"-penta---. Column 16, line k5,

Q r-Q-glycosidically" should read +-O-glycosidically---.

Column 16, line 55, "h-Q-glycosidicall?" should read r- Q-glyco sidically---. Column 16, line 56, "sugar with possesses" should read ---sugar which possesses- Signed and Scaled this thirt Day of January 1976 [SEAL] Arrest:

SUTHI C. MiSON C. MARSHALL DANN 11 (ummr'sxrmrer oj'ParenIs and Trademarks UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION PATENT NO. I DATED April 22, 1975 INVENTOWS) 1 Peter J. L. Daniels It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column t, line 58, "with lead to acetate and," should read ---with lead acetate and,---. Column line +0, 'to about pH." should read ---to about pH Column 6, line 2 "of formula II" should read ---of formula III"--- Column 6, line no, "NRRI 2955" should read ---NRRL 2953---. Column 6, lines 4-8 and &9, "and l, 3,2 ,6", 5" should read ---and l, 5,.

2' ,6 ,5"- Column 7, line 25, "on silica ge" should read on silica gel--- Column T, line 59, 1 7 26 should read -[Ot 7% Column 7, line +1, "FOL 7 26" should read ---[d 7 Column 8, line 1, "IOLY 26 should read "a 7 Column 8, line 15, "[or 7 26 should read F0: 7 Column 8, lines l t and 15, "1,5,?

60' 5-' [a 73 56 130 (water)" should read -l, 5,2' ,6' ,5"-

oz 26 150 (water)---. Column 8 line &8, "chloride a pyrid ne" should read ---chloride in pyridine---. Column 9, line 18, "about ph should read ---about pH Column 9, line 59, "the solution of reflux" should read ---the solution at reflux--- Column 10, line 22, "[aJ 26 should read 1 L73 Column 11, line 6 1 7 26 should read [or 7; Column 16, line 10, "1,5,2' ,6"-penta" should read ---l,5,2',6-penta---. Column 16, line 11, "l,5,2',6"-penta' should read ---l,5,2',6',5"-penta---. Column 16, line k5, "LGL-glycosidically" should read +-O-glycosidically---.

Column 16, line 55, 'W-Q-glycosidicall?" should read +-O-glycosidically--- Column 16, line 56, "sugar with possesses" should read ---sugar which possesses---.

Signed and Sealed this thirteenth a o [SEAL] D y f January 1976 AffeSl.

RUTH C. MASON Arresting Officer C. MARSHALL DANN Commissioner of Parents and Trademarks 

1. THE PROCESS FOR PREPARING A GARAMINE DERIVATIVE HAVING AMINO-PROTECTING GROUPS SELECTED FROM THE GROUP CONSISTING OF LOWER ALKANOYL, AROYL AND ARALKANOYL, WHICH COMPRISES TREATING A PSEUDOTRISACCHARIDE HAVING A GARAMINE MOIETY 4-QGLYCOSIDICALLY LINKED TO ANOTHER PYRANOSIDE SUGAR WHICH POSSESSES AN AMINO FUNCTION AND VICINAL HYDROXYL GROUPS, SAID PSEUDOTRISACCHARIDE HAVING AMINO-PROTECTING GROUPS SELECTED FROM THE GROUP CONSISTING OF LOWER ALKANOYL, AROYL AND ARALKANOYL, WITH A GLYCOL CLEAVING REAGENT SELECTED FROM THE GROUP CONSISTING OF A PERIODATE SALT AND A LEAD (IV) SALT, AND TREATING THE SECO-DIALDEHYDE INTERMEDIATE WITH A HYDRAZINE AND A LOWER, ALKANOIC ACID IN WATER, SAID HYDRAZINE BEING A PHENYLHYDRAZINE.
 1. The process for preparing a garamine derivative having amino-protecting groups selected from the group consisting of lower alkanoyl, aroyl and aralkanoyl, which comprises treating a pseudotrisaccharide having a garamine moiety 4-Q-glycosidically linked to another pyranoside sugar which possesses an amino function and vicinal hydroxyl groups, said pseudotrisaccharide having amino-protecting groups selected from the group consisting of lower alkanoyl, aroyl and aralkanoyl, with a glycol cleaving reagent selected from the group consisting of a periodate salt and a lead (IV) salt; and treating the seco-dialdehyde intermediate with a hydrazine and a lower, alkanoic acid in water, said hydrazine being a phenylhydrazine.
 2. The process of claim 1 wherein said pseudotrisaccharide has a garamine moiety 4-Q-glycosidically linked to another pyranoside sugar with possesses an amino function and vicinal trans diequatorial hydroxyl groups, and wherein said pseudotrisaccharide has amino-protecting groups selected from the group consisting of lower alkanoyl and aroyl having up to 8 carbon atoms.
 3. The process of claim 2 followed by the step of treating the garamine derivative with alkali whereby garamine is formed.
 4. The process of claim 2 for preparing an amino-protected garamine derivative represented by the following structural formula I:
 5. The process of claim 4 wherein R is benzoyl.
 6. The process of claim 4 wherein R is acetyl.
 7. The process of claim 4 wherein said glycol cleaving reagent is an alkali metal meta-periodate, said hydrazine is phenylhydrazine, and said lower alkanoic acid is acetic acid.
 8. The process of claim 4 wherein said pseudotrisaccharide is a compound of formula II or a mixture thereof, wherein R is acetyl; said glycol cleaving reagent is sodium meta-periodate; said hydrazine is phenylhydrazine, and said lower alkanoic acid is acetic acid, whereBy is formed 1,3,3''-tri-N-acetylgaramine.
 9. The process of claim 8 wherein said pseudotrisaccharide represented by formula II is 1,3,2'',3''''-tetra-N-acetylantibiotic G-418.
 10. The process of claim 8 wherein said pseudotrisaccharide represented by formula II is 1,3,6'',3''''-tetra-N-acetylgentamicin B1.
 11. The process of claim 8 wherein said pseudotrisaccharide starting compound is a mixture comprising 1,3,2'',6'',3''''-penta-N-acetylantibiotic JI-20A and 1,3,2'',6'',3''''-penta-N-acetylantibiotic JI-20B.
 12. The process of claim 8 wherein said pseudotrisaccharide represented by formula II is 1,3,2'',6'',3''''-penta-N-acetylantibiotic JI-20B.
 13. The process of claim 8 wherein said pseudotrisaccharide starting compound is a mixture comprising 1,3,6'',3''''-tetra-N-acetylgentamicin B, 1,3,6'',3''''-tetra-N-acetylgentamicin B1 and 1, 3,2'',3''''-tetra-N-acetylgentamicin X2. 